Variation or polymorphism in a genome commonly exists in biological organisms; it mainly refers to differences of genomic sequences among different species or among different individuals of the same species, including differences of sequences in the gene coding region and non-coding region. Variation or polymorphism of DNA reflects the evolutionary process of the establishment, selection, migration, recombination and mating system of a species, laying the foundation of the rich and colorful biological world, and it serves as the basis of biodiversity. The most common and simple form of DNA variation or polymorphism is the variation or polymorphism of a single nucleotide in the genome, namely single nucleotide variation or polymorphism. Single nucleotide polymorphism (or SNP) is the most commonly seen form of variation in human genomic DNA sequence, and is one important genetic marker, which is believed to be associated with an individual's phenotypic differences, susceptibility to a disease, and response or resistance to a drug, etc. Such a variation or polymorphism also widely exists in other species, and has important biological functions as well. Thus, the detection of single nucleotide variation or polymorphism is of great significance.
There are many methods useful for detecting and genotyping single nucleotide variation (or SNP) (Ragoussis, J. Annu Rev Genomics Hum Genet, 2009, 10, 117-133), such as restrictive fragment length polymorphism, single strand conformation polymorphism analysis, allele specific oligonucleotide probe hybridization, denatured gradient gel electrophoresis, allele specific amplification system, oligonucleotide ligation analysis, reverse dot blot, denaturing high performance liquid chromatography, mass spectrometry, gene chips, pyrosequencing, gene sequencing and so on. Since all these technologies require post PCR manipulations, it is easy to cause contamination of the amplification products. The analysis procedure is relatively complicated and time-consuming, and cannot meet the clinical requirement of being fast and convenient.
Real-time PCR refers to carrying out amplification and detection simultaneously, wherein the amplification process is indicated through detecting changes of fluorescence signals in amplification cycles. As an important homogenous detection technology, recently, real-time PCR has been widely used in the detection and genotyping of single nucleotide variation or SNP. Currently, based on whether a fluorescent probe is used, real-time PCR can be divided into the probe-based type and the non-probe-based type. Since the probe-based real-time PCR has probe that can increase the specificity in recognizing a template, it is more specific than the detection results from the non-probe-based real-time PCR. The probe-based real-time PCR can also achieve the aim of detecting multiple target sequences by labeling different target-specific probes with different fluorescent groups. Thus, the probe-based real-time PCR is relatively more commonly used. There are many types of probes used in the probe-based real-time PCR, including TaqMan™ probe (Livak, K. J. Genetic Analysis, 1999, 14, 143-149.), TaqMan-MGB™ probe (Afonina, I. A. et al, Biotechniques, 2002, 32, 4, 940-944, 946-949), molecular beacons (Tyagi, S. et al, Nature Biotechnology, 1998, 16, 49-53), displacing probe (Li, Q., et al, Nucleic Acids Research, 2002, 30, E5.), scorpions (Whitcombe, D. et al, Nature Biotechnology, 1999, 17, 804-807), amplifier primer (Nazarenko, I. A., et al, Nucleic Acids Research, 1997, 25, 1516-1521) etc. When these probes are used in the detection of single nucleotide variation or SNP, the most often used mode is that two allele-specific probes labeled with different fluorescent groups are used to detect a single nucleotide variation or SNP, as described by Ruan L et al. (Ruan, L., et al. Transfusion. 2007, 47(9):1637-42), wherein they accomplished the detection of 6 single nucleotide variations or SNPs in 3 reaction tubes. Restricted by the number of detection channel of the existing real-time PCR machines, using said method, at most 3 single nucleotide variations or SNPs could be detected simultaneously in a single tube. Therefore, although real-time PCR can meet the clinical requirements of being fast and convenient, however, the number of single nucleotide variations or SNPs detected in a single tube is limited, and it often needs several tubes to detect multiple single nucleotide variations or SNPs, which fails to meet the high throughput requirement in clinical testing, and the cost is relatively high.
Another approach of detecting single nucleotide variation or SNP is using melting curve analysis after real-time PCR, wherein the genotype of a single nucleotide variation or SNP is identified by differences of the melting temperature when a probe hybridizes with different targets. Using this detection approach, genotyping of one single nucleotide variation or SNP can be achieved using one fluorescent probe. In comparison with the real-time PCR approach discussed above (wherein the genotype of one single nucleotide variation or SNP is detected by using two probes labeled with different colors), the number of single nucleotide variations or SNPs detected using this method in a single tube is doubled. As described by Nicklas J A et al. (Nicklas, J. A., et al. Journal of Forensic Sciences, 2008, 53(6):1316-24), wherein they achieved with a single tube the detection of the genotype of 6 single nucleotide variations or SNPs in a 6 color real-time PCR machine, using melting curve analysis by adjacent probes. However, for detecting more single nucleotide variations or SNPs, this method still needs more reaction tubes, and thus still cannot meet the clinical requirement of high throughput detection. In addition, because only one or few single nucleotide variations or SNPs can be detected using one fluorescent probe, the cost is still relatively high.